Ferroportin (Fpn, IREG1, MTP1) is the only identified transmembrane iron exporter in vertebrates. Our preliminary data indicate that hepcidin inhibits cellular iron export by inducing the internalization and degradation of Fpn. The goals of this proposal are to understand the mechanism of Fpn-mediated iron export and the mechanism of hepcidin-mediated Fpn internalization. The entry of iron into plasma is highly regulated and is affected by iron stores, inflammation and hypoxia. Chronic inflammation results in decreased plasma iron and an iron-limited anemia, referred to as the Anemia of Chronic Disease. Increased iron stores or inflammation result in high levels of hepcidin, a peptide secreted by liver that regulates iron metabolism. We hypothesize that this interaction is a major controlling factor in mammalian iron homeostasis. We will determine if metal export by Fpn is specific for iron through the use of metal sensitive fluorescent dyes. We will define the structure of Fpn and determine the number of transmembrane domains and the location of intracellular and extracellular domains. Biochemical and genetic approaches will be used to determine how hepcidin binds to Fpn and how Fpn is internalized. Human hepcidin will be modified to generate radiolabeled and photoaffinity ligands, which will then be used to characterize the hepcidin/Fpn interaction. By selectively changing amino acid residues in human and fish hepcidins, we will define residues critical for Fpn binding. Residues in Fpn that are required for internalization and metal transport will be identified through genetic screens and biochemical examination of hepcidin treated Fpn. RNAi silencing will be used to determine if Fpn is the only mammalian iron exporter or if are there are other iron export systems. These studies will probe the mechanism that underlines the regulation of iron export by hepcidin and will define the roles of hepcidin and Fpn in iron physiology.